SUMMARY/ABSTRACT (PROJECT 1) Cellular immunotherapy has revolutionized the treatment of a variety of cancers. Anti-CD19 chimeric antigen receptor redirected T cells (CART19) have been particularly successful in B cell malignancies, with complete remissions as high as 80-90% in certain types of advanced leukemia. Many of these remissions are sustained, but the major limitation is relapse in 20-50% of patients, and two-thirds of these relapses involve antigen escape. There are also numerous patients with hematologic malignancies who cannot benefit from this therapy because of the inability to collect or manufacture T cells, especially from intensively treated subjects with low counts of T cells that are defective at baseline. Additional resistance to CAR T cell therapy, particularly in solid tumors, is multifactorial and the causes of treatment failure include antigen heterogeneity, poor trafficking, soluble inhibitory factors present in the tumor microenvironment, overexpression of negative immune checkpoint molecules as well as suppression due to intrinsic inhibitory T cell programs that may be epigenetically regulated. Notwithstanding these challenges, even when CAR T cells elicit potent anti-tumor effects, the success of this approach is often hampered by dose-limiting immunotoxicities, including cytokine release syndrome (CRS) that can cause death in patients. This project will establish a comprehensive strategy to target the major limitations in CAR T cell therapy, increasing the feasibility of this already transformational approach. In Aim 1, we will use an innovative genetic knock-in approach to express multiple CARs (i.e., a ?CAR fleet?) in T cells, while simultaneously disrupting negative regulators of proliferation and anti-tumor function. The goal here is to employ genetic and cellular engineering principles to design more potent CAR T cells resistant to intrinsic and extrinsic dysfunction. In Aim 2, we will engineer CAR T cells to constitutively or inducibly produce inhibitors of catecholamines, which were recently discovered to be central regulators of CAR T cell-induced CRS. These studies will determine whether engineered production of soluble atrial natriuretic peptides (ANPs) by CAR T cells reduces systemic ?CRS-like? immunotoxicity and improves the anti-tumor responses mediated by transferred T cells. Finally in Aim 3, we will exploit optofluidic technology to develop a robust biomanufacturing process for isolating purely ?fit,? highly- proliferative anti-cancer T cell subsets. The long-term objective of this work is to create a more effective and uniform clinical CAR T cell infusion products at lower costs. This project is expected to open therapeutic horizons in the field of adoptive immunotherapy and offer new research prospects that could be translated to improving treatment of many different cancers.